The -amyloid deposits found in the brains of patients with Alzheimer's disease are composed of peptides (A) derived by proteolytic cleavage of the amyloid precursor protein (APP), ()a glycoprotein with a large extracellular domain, a single transmembrane region, and a short cytoplasmic tail. A portion of cell-associated APP is normally cleaved within its extracellular domain by an uncharacterized protease known as -secretase. This process releases a larger soluble APP fragment (APPs) into the extracellular space (Weidemann et al., 1989), and, because it cleaves APP within the A domain, it does not generate amyloid (Esch et al., 1990; Sisodia et al., 1990; Anderson et al., 1991; Wang et al., 1991). A number of alternative metabolic pathways have also been described that have the potential to generate intact A fragments (Estus et al., 1992; Golde et al., 1992; Haass et al., 1992a; 1992b; Seubert et al., 1992; 1993; Shoji et al., 1992). Secretory cleavage of APP is increased by phorbol esters (Caporaso et al., 1992; Gillespie et al., 1992; Slack et al., 1993), which are potent activators of protein kinase C (PKC), and also by binding of receptor ligands (such as carbachol, interleukin 1, and thrombin) to specific cell surface receptors (Van Nostrand et al., 1990; Buxbaum et al., 1992; Nitsch et al., 1992). In human embryonic kidney (HEK) cell lines stably transfected with individual muscarinic receptor subtypes (Peralta et al., 1988), activation by carbachol of m1 and m3, but not m2 and m4 receptor subtypes increased APPs release (Nitsch et al., 1992). Stimulation of APPs release from m1 receptor-expressing HEK cells by carbachol or phorbol esters was accompanied by a decrease in the release of A fragments (Hung et al., 1993), suggesting that these agents activate a pathway that cleaves APP within the A domain and hence might prevent amyloid formation, although in some cell types, APPs secretion and A formation appear to be independently regulated (Gabuzda et al., 1993). In view of the possible reciprocal relationship between APPs secretion and A generation, as well as the putative role of APPs as a neurotrophic, neuroprotective agent (Milward et al., 1992; Mattson et al., 1993), it is important that the factors regulating the secretory processing of APP be fully understood.

Muscarinic m1 and m3 receptors, which stimulate APPs release when activated, are also efficiently coupled to phosphoinositide (PI) hydrolysis in HEK cells, while m2 and m4 receptors are not (Peralta et al., 1988; Sandmann et al., 1991). Hydrolysis of PI generates diacylglycerol, an activator of protein kinase C (Nishizuka, 1992), implicating this enzyme in the stimulation of APPs formation by muscarinic agonists. This hypothesis is supported by evidence that APP is directly phosphorylated by PKC in vitro (Gandy et al., 1988; Suzuki et al., 1992). However, mutation of serine and threonine residues in the cytoplasmic tail of the APP molecule, or deletion of the entire cytoplasmic domain, failed to suppress the stimulation of APPs release by phorbol esters (da Cruz e Silva et al., 1993), suggesting that direct phosphorylation of amino acid residues in the intracellular domain of mature APP is not the mechanism responsible for the enhancement of APPs formation.

Muscarinic receptors are coupled to effector proteins via heterotrimeric guanine nucleotide-binding proteins (G-proteins) (Ashkenazi et al., 1987; 1989). PI-coupled muscarinic receptors stimulate the -isozyme of PI-specific phospholipase C by activating G-proteins of the G class (Blank et al., 1991; Taylor et al., 1991; Berstein et al., 1992). However, activation of transfected muscarinic m5 receptors, but not m2 receptors, was recently shown to stimulate PI turnover in part via increased tyrosine phosphorylation of the -isozyme of phospholipase C (phospholipase C) (Gusovsky et al., 1993). Moreover, stimulation of muscarinic receptors coupled to PI hydrolysis increased tyrosine phosphorylation in the hippocampus (Stratton et al., 1989), and activation of muscarinic m1 receptors suppressed potassium channel activity via a tyrosine kinase-dependent mechanism (Huang et al., 1993). These results implicate tyrosine phosphorylation both as a cause and a possible consequence of muscarinic receptor-mediated PI hydrolysis.

We tested the possibility that tyrosine phosphorylation regulates APP processing in cultured HEK cells stably expressing different muscarinic receptor subtypes. Our findings suggest that a tyrosine phosphorylation-dependent mechanism contributes to the stimulation of APP secretory cleavage by muscarinic agonists acting on m1 and m3 receptors and by direct activators of PKC. In addition, we show that the increase in tyrosine phosphorylation induced by inhibiting enzymes (tyrosine phosphatases) that dephosphorylate protein tyrosine residues is sufficient to stimulate APPs release.

EXPERIMENTAL PROCEDURES

Materials

Cell Culture

Measurement of APPs Release

Immunoprecipitation and Measurement of Anti-phosphotyrosine Immunoreactive Proteins

Inositol Phosphate Formation

Statistical Analysis

RESULTS

As reported previously (Nitsch et al., 1992), exposure to the cholinergic agonist carbachol significantly increased the release of APPs from HEK cells transfected with m3 muscarinic receptors (Fig. 1). Stimulation of these receptors triggers PI hydrolysis (Peralta et al., 1988; Sandmann et al., 1991) and increases levels of the endogenous PKC activators diacylglycerol and calcium (Nishizuka, 1992; Berridge, 1993). Knowledge of these relationships, together with the observation that the protein kinase inhibitor staurosporine inhibited carbachol-induced APPs release, implicated protein kinase C in the APPs response. Consistent with this hypothesis, PMA, a potent, nonphysiological activator of PKC, was able to increase APPs release as effectively as carbachol (Fig. 1). However, treatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (Fantus et al., 1989; Heffetz et al., 1990) also increased APPs release (Fig. 1), and sodium orthovanadate, a less potent tyrosine phosphatase inhibitor (Trudel et al., 1991), exerted a modest, concentration-dependent stimulatory effect as well. These results suggested that an increase in the phosphorylation of tyrosine, as well as in the PKC-dependent phosphorylation of serine or threonine, can enhance APP processing. Moreover, because staurosporine, used in previous studies as an inhibitor of PKC (Nitsch et al., 1992), is in fact an effective inhibitor of both protein kinase C (Tamaoki et al., 1986) and protein tyrosine kinases (Rüegg and Burgess, 1989; Shiseva and Shechter, 1993), use of this drug may not distinguish between effects that are dependent on PKC and those due to tyrosine phosphorylation.

Figure 1: Release of APPs is stimulated by treatments that increase PKC activity or protein tyrosine phosphorylation. A, HEK cells stably expressing m3 muscarinic receptors were incubated in serum-free control medium (con) or in medium containing carbachol (carb), sodium orthovanadate (van), pervanadate (pvan), or PMA (pma) for 1 h. APPs released into the medium was measured by immunoblot as described under ``Experimental Procedures.'' Results are expressed as means ± S.E. of three to five experiments performed in triplicate. *, significantly different from the control group, p < 0.05 by analysis of variance. B, immunoblot showing APPs in medium from HEK m3-expressing cells following a 1-h incubation in serum-free control medium (con) or medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM).

Carbachol stimulated APPs release to a significantly greater extent in cells expressing m3 than m2 muscarinic receptor subtypes (Fig. 2A), as described previously (Nitsch et al., 1992). In contrast, PMA and pervanadate stimulated APPs secretion equally effectively in both cell lines (Fig. 2A). In addition to stimulating APPs release, these agonists also increased protein tyrosine phosphorylation but to varying degrees. Tyrosine phosphorylation was assessed by immunoprecipitating tyrosine-phosphorylated proteins from lysates of control and treated cell cultures and then preparing immunoblots of the precipitates with antiphosphotyrosine antibodies. In cells expressing m3 receptors, carbachol could be shown to increase tyrosine phosphorylation of at least two proteins (appearing in some experiments as two doublets) with approximate molecular masses ranging from 70 to 110 kDa (Fig. 2B). The fastest migrating (70 kDa) band was the one most affected by carbachol. PMA elicited a similar pattern of tyrosine phosphorylation, although the effect on the 70-kDa protein was smaller than that of carbachol (Fig. 2C). In contrast, pervanadate treatment increased tyrosine phosphorylation of a large number of proteins (Fig. 2C). Co-incubation of the antiphosphotyrosine antibodies with 5 mM phospho-L-tyrosine prevented the appearance of these bands, confirming the specificity of the antibodies (data not shown). (Note that the separation of the bands in Fig. 2C was increased relative to those in Fig. 2B by prolonging the electrophoresis time.)

Figure 2: Stimulation of APPs release and tyrosine phosphorylation by carbachol is more pronounced in m3- than in m2-expressing HEK cells. A, HEK cells expressing m3 (openbars) or m2 (hatchedbars) muscarinic receptors were treated with carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM) for 1 h and medium APPs content was measured by immunoblot. Results are expressed as means ± S.E. from four to five experiments, except for pervanadate treatment values, which are means of two experiments. *, significantly different from corresponding treatment in m3-transfected cells, p < 0.05 by paired t test. B, Immunoblot of tyrosine-phosphorylated proteins in anti-phosphotyrosine immunoprecipitates from HEK cells expressing m3 (lanes1-4) or m2 (lanes5-8) muscarinic receptors. Cells were preincubated for 15 min in serum-free medium and then treated for 10 min in fresh medium containing varying concentrations of carbachol (carb). C, immunoblot of tyrosine-phosphorylated proteins in anti-phosphotyrosine immunoprecipitates from m3- (lanes1-4) and m2- (lanes5-8) expressing HEK cells. Cultures were preincubated for 15 min in serum-free medium and then treated for 10 min in fresh control serum-free medium, or in medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM). Note that in C, the bands are more widely separated than in B because electrophoresis was continued for a longer period of time.

Carbachol-stimulated tyrosine phosphorylation was much less pronounced in cells expressing m2 receptors than in m3-expressing cells (Fig. 2B). This effect was consistently observed in four separate experiments. PMA and pervanadate exerted similar effects in both lines (Fig. 2C). Similarly, carbachol-stimulated tyrosine phosphorylation to a greater extent in m1- than in m4-expressing cells (data not shown). Examination of the time course of carbachol-stimulated tyrosine phosphorylation in m3-expressing cells demonstrated that protein phosphorylation was maximal within 10 min and remained stable for at least 30 min (not shown). This rise preceded the increase in APPs release, which reached half-maximal levels within 10 min and was maximal by 30 min (Nitsch et al., 1992).

The results showed a correlation between stimulation of APPs release and enhanced tyrosine phosphorylation. Accordingly, we examined the effects on these responses of the tyrosine kinase inhibitors tyrphostin A25, lavendustin A, and genistein. Overnight pretreatment with tyrphostin A25 (100 µM) reduced the effects of carbachol and pervanadate on APPs release by 54 ± 11 and 49 ± 4%, respectively, and inhibited PMA's effect by 37 ± 8% (Fig. 3A). Concomitantly, tyrphostin caused a significant overall reduction in pervanadate-induced tyrosine phosphorylation (by 34 ± 4%, n = 3) (Fig. 3B) and significantly reduced carbachol-mediated phosphorylation of the 70 kDa band by 41 ± 7% (n = 4) (Fig. 3C). PMA-induced phosphorylation was not significantly reduced. Lavendustin A (100 µM) did not affect APPs release elicited by carbachol, PMA, or pervanadate (data not shown).

Figure 3: Tyrphostin A25 inhibits evoked APPs release and tyrosine phosphorylation. A, HEK cells expressing m3 muscarinic receptors were pretreated for 18 h with dimethyl sulfoxide (vehicle control; openbars) or 100 µM tyrphostin A25 (hatchedbars) in serum-free medium and then incubated for 1 h in fresh control medium or in medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM). APPs released into the medium during a 1-h incubation period was measured by immunoblot. Results are expressed as means ± S.E. from three to eight experiments. *, significantly different from corresponding cultures incubated in the absence of antagonist, p < 0.05 by paired t test. B, immunoblot of tyrosine-phosphorylated proteins in anti-phosphotyrosine immunoprecipitates from m3 receptor-expressing HEK cells treated for 10 min with control medium or medium containing pervanadate (pvan, 250 µM). Cells were pretreated for 18 h in serum-free medium containing 0.1% dimethyl sulfoxide (vehicle control) or tyrphostin A25 (100 µM). C, immunoblot of tyrosine-phosphorylated proteins in cells exposed to control medium or medium containing carbachol (carb, 100 µM) for 10 min. Cells were pretreated with dimethyl sulfoxide or tyrphostin A25 as in B.

The broad spectrum tyrosine kinase inhibitor genistein (Akiyama et al., 1987; Levitzki, 1992) caused a dose-dependent inhibition in both basal and carbachol-induced release of APPs from HEK cells transfected with m3 muscarinic receptors. Inhibition was significant only at high concentrations (100 µg/ml genistein), which also blocked the responses to PMA and to pervanadate (Fig. 4, A and B). This concentration of genistein caused a concomitant reduction in resting levels of tyrosine phosphorylation, and, in three to four independent experiments, it inhibited the increase in tyrosine phosphorylation elicited by carbachol, PMA, and pervanadate by 69 ± 9, 87 ± 4, and 62 ± 6%, respectively (Fig. 4C). Comparable concentrations of genistein were required to maximally inhibit tyrosine phosphorylation of the EGF receptor by EGF in intact A431 cells (Akiyama et al., 1987), or to block activity of a cytosolic tyrosine kinase in rat adipocytes (Shisheva and Shecter, 1993).

Figure 4: Genistein inhibits evoked APPs release and tyrosine phosphorylation. A, HEK cells expressing m3 muscarinic receptors were treated for 1 h in serum-free control medium or in medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM) or pervanadate (pvan, 250 µM) in the absence (openbars) or presence (hatchedbars) of 100 µg/ml genistein. APPs released into the medium was measured by immunoblot. Results are expressed as means ± S.E. from three to five experiments. *, significantly different from corresponding cultures incubated in the absence of genistein, p < 0.05 by paired t test. B, immunoblot of APPs released into the medium from m3-expressing HEK cells. Treatments were as described in A. C, immunoblot of tyrosine-phosphorylated proteins in anti-phosphotyrosine immunoprecipitates from cells pretreated for 30 min with genistein or vehicle (0.2% dimethyl sulfoxide) in serum-free medium and then treated for 10 min with agonists in the presence or absence of genistein as in A.

Previous studies indicated that the protein kinase inhibitor staurosporine inhibited APPs release in response to carbachol (Nitsch et al., 1992). However, the bisindolylmaleimide GF 109203X, which selectively inhibits PKC without affecting tyrosine kinase activity (Toullec et al., 1991), completely blocked PMA-induced APPs release but reduced the effect of carbachol by only 40% and did not affect pervanadate-stimulated APPs release at all (Fig. 5A). This inhibitor reduced carbachol-mediated tyrosine phosphorylation of the 70 kDa band by an average of 53 ± 7% (n = 3), while reducing PMA-induced phosphorylation by 68 ± 9% (n = 4) (Fig. 5B). It did not affect pervanadate-mediated phosphorylation, suggesting that pervanadate's effect on APPs release is independent of PKC. However, because orthovanadate has been shown to stimulate phospholipase C activity and PI turnover in permeabilized mast cells (Atkinson et al., 1993), the possibility that pervanadate might stimulate PI turnover by a similar mechanism in HEK cells was tested. Pervanadate (250 µM) increased tyrosine phosphorylation of several proteins found in anti-phospholipase C immunoprecipitates. The most prominent band had an apparent molecular mass of 150 kDa (Fig. 6A, arrow), tentatively identifying it as phospholipase C (Rhee, 1991). Additional bands found in the immunoprecipitates may represent associated proteins. Bands were detectable only after prolonged exposure of the film and were not observed in immunoprecipitates from cultures exposed to PMA, carbachol, or control medium. The effect of pervanadate was abolished by genistein. Although tyrosine phosphorylation of phospholipase C stimulates its activity (Rhee, 1991), pervanadate treatment elicited only a modest (1.3-fold) increase in inositol phosphate formation (Fig. 6B); the mean increase observed in three experiments was not statistically significant. In contrast, carbachol (100 µM) increased inositol phosphate formation by 7.0-fold (Fig. 6B).

Figure 5: Effect of GF 109203X on evoked APPs release and tyrosine phosphorylation. A, HEK cells expressing m3 receptors were treated for 1 h in serum-free control medium or in medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM) in the absence (openbars) or presence (hatchedbars) of 2.5 µM GF 109203X. Results are expressed as means ± S.E. from five to eight experiments. *, significantly different from corresponding cultures incubated in the absence of GF 109203X, p < 0.05 by paired t test. B, immunoblot of tyrosine-phosphorylated proteins in anti-phosphotyrosine immunoprecipitates from cells pretreated for 15 min in serum-free medium containing vehicle (0.05% dimethyl sulfoxide) or GF 109203X and then treated for 10 min with agonists in the presence or absence of GF 109203X as in A.

Figure 6: Pervanadate increases tyrosine phosphorylation of phospholipase C, but not inositol phosphate formation. A, HEK cells expressing m3 receptors were pretreated for 30 min with serum-free medium containing genistein (100 µg/ml), or 0.2% dimethyl sulfoxide and then treated for 10 min with control medium (con) or medium containing carbachol (carb, 100 µM), PMA (pma, 1 µM), or pervanadate (pvan, 250 µM). Proteins immunoprecipitated with anti-phospholipase C antiserum were immunoblotted with antiphosphotyrosine antibodies. B, formation of total IPs was measured in cells exposed for 1 h to carbachol (100 µM) or pervanadate (250 µM). Results are expressed as means ± S.E. from three separate experiments performed in quadruplicate. *, significantly different from control cultures, p < 0.05 by analysis of variance.

The association between APPs release and tyrosine phosphorylation raised the possibility that APP processing might be regulated via direct phosphorylation of a tyrosine residue in the cytoplasmic domain of the molecule. However, when antiphosphotyrosine immunoprecipitates were separated on SDS gels and immunoblots were prepared using an antibody to APP, no bands were detected (data not shown), suggesting that cell-associated APP was not directly phosphorylated by any of the treatments that stimulate its secretory processing. This is consistent with reports that deletion of the cyplasmic tail of APP fails to prevent the stimulation of APPs release by phorbol esters (da Cruz e Silva et al., 1993).

DISCUSSION

These data suggest that protein tyrosine phosphorylation contributes to the stimulation of APPs release by muscarinic receptor activation in HEK cells. Thus, treatment of m1 and m3 muscarinic receptor-expressing HEK cells with carbachol increased APPs release and tyrosine phosphorylation in parallel, with similar time course and dose-response characteristics. Both effects of carbachol were considerably attenuated in cells expressing m2 and m4 muscarinic receptor subtypes (Nitsch et al., 1992; Fig. 2; and data not shown), suggesting that tyrosine phosphorylation, like PI turnover and APPs release (Peralta et al., 1988; Sandmann et al., 1991; Nitsch et al., 1992), is preferentially coupled to m1 and m3 receptor subtypes. The stimulation of APPs release by carbachol was blocked by high concentrations of the broad spectrum tyrosine kinase antagonist genistein and was significantly reduced by tyrphostin A25. Moreover, the tyrosine phosphatase inhibitor pervanadate mimicked the effect of carbachol on APPs release. These results implicate a tyrosine phosphorylation-dependent step in the stimulation of APPs release by carbachol, and, potentially, by other receptor agonists as well. Because stimulation of APPs release is accompanied by increased cleavage of APP within the A domain of APP (Hung et al., 1993), activation of the relevant signal transduction pathways may represent a mechanism to reduce the formation of amyloid. (It is important to note that the 22C11 antibody used in this study also recognizes members of the APP-like protein (APLP) family (Wasco et al., 1992; 1993; Slunt et al., 1994). APLPs are also expressed at high levels in mouse and human brain, but lack the extracellular 28 residues of the A sequence. However, blots probed with the antibody R1736 (Haass et al., 1992b; 1994), which is specific for APP, showed a pattern of APPs release in response to PMA, carbachol, and pervanadate that was very similar to that obtained using the 22C11 antibody (data not shown). The possible contribution of APLPs to the secretory responses of HEK cells remains to be determined.

The role of PKC in carbachol-mediated APPs release was reexamined using the specific PKC antagonist GF 109203X. The blockade of carbachol-mediated stimulation of APPs release by the protein kinase inhibitor staurosporine (Nitsch et al., 1992) is difficult to interpret because this compound inhibits both PKC (Tamaoki et al., 1986) and tyrosine kinases (Bourgoin and Grinstein, 1992; Shisheva and Schecter, 1993). The bisindolylmaleimide GF 109203X, however, inhibits PKC while sparing tyrosine kinases (Toullec et al., 1991), and indeed, this compound reduced carbachol-mediated stimulation of APPs release by only 40%, while nearly abolishing the effect of PMA (Fig. 5A). Similarly, Buxbaum et al.(1994) reported that prior down-regulation of PKC in m3-transfected Chinese hamster ovary cells did not affect the stimulation of APPs release by the muscarinic agonist bethanechol, but blocked the effect of acutely administered phorbol esters. Carbachol-mediated tyrosine phosphorylation in HEK cells was also reduced but not abolished by GF 109203X, suggesting that both PKC-dependent and PKC-independent pathways contribute to the increased tyrosine phosphorylation and APPs release evoked by muscarinic receptor stimulation. The putative signaling pathways involved in regulation of APPs release in HEK cells are summarized in Fig. 7.

Figure 7: APPs release from HEK cells expressing m3 muscarinic receptors is regulated by PKC- and tyrosine phosphorylation-dependent signal transduction pathways. Activation of muscarinic m1 or m3 receptors (either by the neurotransmitter acetylcholine (ACh) or by the cholinergic agonist carbachol) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP) and activates PKC by generating diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP). The latter releases calcium (Ca), required for the activity of group A PKCs. PMA and carbachol stimulate tyrosine phosphorylation and APPs release, but while the effects of PMA are almost entirely blocked by the specific PKC inhibitor GF 109203X, carbachol acts in a partially PKC-independent manner. The pathways activated by both carbachol and PMA appear to converge at a tyrosine phosphorylation-dependent step; they are partially reduced by tyrphostin A25, and nearly abolished by genistein. Because genistein is known to exert nonspecific inhibitory effects, the possible participation of a tyrosine phosphorylation-independent component cannot be ruled out (see brokenarrow). Pervanadate increases tyrosine phosphorylation, presumably via inhibition of protein tyrosine phosphatases. Its effect on APPs release is not inhibited by GF 109203X, suggesting that it acts independently of PKC. Stimulatory effects are indicated by closedarrows; and inhibitory effects are indicated by openarrows. Pathways denoted by twoarrows indicate potentially multi-step processes.

In HEK cells expressing m3 receptors, PMA stimulated APPs release as effectively as carbachol and induced a similar, although less pronounced, pattern of tyrosine phosphorylation. The effect of PMA on both parameters was almost completely blocked by GF 109203X, indicating that both effects are mediated by PKC.

The tyrosine kinase inhibitor tyrphostin A25 reduced carbachol- and PMA-induced APPs release by 54 and 37%, respectively, while genistein abolished the responses to both agents. This difference might reflect the broader specificity of genistein and suggests that APPs release may be regulated by one or more tyrosine kinases with differential sensitivity to these antagonists. Although the results implicate tyrosine phosphorylation in the stimulation of APPs release by both carbachol and PMA, as well as by pervanadate, it is important to recognize that genistein has a variety of additional effects, including inhibition of protein synthesis (Hu et al., 1993); thus, results obtained with this inhibitor must be interpreted with caution. Despite this caveat, the data, when taken together, strongly support a role for increased tyrosine phosphorylation in the regulation of APPs release in HEK cells. Although the mechanism by which PMA and carbachol increase tyrosine phosphorylation was not addressed in this study, others have shown that PMA potentiates the inhibition of tyrosine phosphatase activity by sodium orthovanadate in macrophages (Zor et al., 1993). It is possible that a similar inhibitory effect accounts for the effects of carbachol and PMA observed here. Alternatively, these compounds might directly stimulate tyrosine kinase activity.

The protein tyrosine phosphatase inhibitor pervanadate stimulated APPs release and increased protein tyrosine phosphorylation. Pervanadate can be generated in vitro by combining equimolar amounts of sodium orthovanadate and HO (Fantus et al., 1989; Trudel et al., 1991); it is a potent and cell-permeant inhibitor of tyrosine phosphatases and possibly an activator of tyrosine kinases as well (Trudel et al., 1991; Zor et al., 1993). Pervanadate increased tyrosine phosphorylation of a 150-kDa protein immunoprecipitated by an antibody against phospholipase C, but it did not significantly elevate inositol phosphate formation, possibly because the tyrosine residues phosphorylated were not those involved in regulation of catalytic activity. Moreover, the stimulation of APPs release by pervanadate was not inhibited by GF 109203X. Zor et al. (1993) showed that pervanadate activates phospholipase A, an enzyme previously implicated in the stimulation of APPs release by carbachol in cells transfected with muscarinic m1 receptors (Emmerling et al., 1993). However, whereas inhibition of PKC by GF 109203X blocked pervanadate-induced phospholipase A activation, GF 109203X did not affect pervanadate-induced tyrosine phosphorylation, either in macrophages (Zor et al., 1993) or in the HEK cells used in the present study. The evidence overall indicates that, whereas pervanadate may be capable of eliciting PKC-dependent effects in some cell types, the responses to pervanadate observed in the present study did not involve PKC activation. In contrast, both pervanadate-evoked APPs release and tyrosine phosphorylation were sensitive to the tyrosine kinase inhibitors genistein and tyrphostin A25. Although the cytoplasmic domain of APP contains a number of potential phosphorylation sites for both PKC and tyrosine kinases and can be phosphorylated on serine by PKC under in vitro conditions (Gandy et al., 1988; Suzuki et al., 1992), deletion of the cytoplasmic domain did not affect the increase in APPs release elicited by phorbol esters (da Cruz e Silva et al., 1993), suggesting that the kinases mediating secretory cleavage of APP have as their targets proteins other than APP. Similarly, our results suggest that direct phosphorylation of APP on tyrosine is not associated with stimulation of its release because we were unable to immunoprecipitate cell-associated APP from either control or stimulated cells with an anti-phosphotyrosine antibody .

Activation of m5 but not m2 receptors in transfected Chinese hamster ovary cells was recently shown to stimulate tyrosine phosphorylation of phospholipase C (Gusovsky et al., 1993), an isozyme known to couple receptor tyrosine kinases to PI hydrolysis (Kim et al., 1991). Phosphorylation of phospholipase C by carbachol in m5 receptor-expressing cells was secondary to activation of receptor-operated calcium channels (Gusovsky et al., 1993). In the present study, although carbachol increased tyrosine phosphorylation of several proteins, none of these corresponded in size to phospholipase C (145-148 kDa), and no phosphorylated proteins were observed in anti-phospholipase C immunoprecipitates from carbachol-treated cells, suggesting that other proteins mediate the increase in APPs release observed in HEK cells. At present, the identities of these putative intermediates in the APP secretory pathway are unknown. Other G-protein-coupled receptors that stimulate tyrosine kinases include the bombesin, bradykinin, vasopressin, endothelin, and cholecystokinin receptors (Zachary et al., 1991, 1992; Leeb-Lundberg and Song, 1991; Lutz et al., 1993). Moreover, direct activation of PKC by phorbol esters increases protein tyrosine phosphorylation in fibroblasts (Bishop et al., 1983; Gilmore and Martin, 1983; Cooper et al., 1984; Zachary et al., 1991), in pancreatic acinar cells (Lutz et al., 1993), and in PC12 cells (Maher et al., 1988; Thomas et al., 1992).

Abnormalities in protein tyrosine kinases have been described in Alzheimer's disease; these include decreased tyrosine kinase activity in particulate fractions from frontal cortex (Shapiro et al., 1991) and hippocampus (Vener et al., 1993) and increased levels of several cortical cytosolic antiphosphotyrosine immunoreactive peptides (Shapiro et al., 1991). The latter could be the result of reduced tyrosine phosphatase activity. No alterations were detected in membrane-associated antiphosphotyrosine immunoreactive peptides in hippocampus (Vener et al., 1993) or in cortex (Shapiro et al., 1991). These observations, together with the evidence presented in the current study, raise the possibility that abnormal reductions in protein tyrosine kinase activity in Alzheimer's disease may result in reduced secretory processing of APP. This could increase the formation of amyloidogenic fragments and impair the growth-promoting and neuroprotective function of APPs (Saitoh et al., 1989; Milward et al., 1992; Roch et al., 1992; Mattson et al., 1993) and possibly of APLPs. Moreover, these data may have implications for the processes governing the proteolytic conversion of a large number of transmembrane growth factors and receptors into diffusible molecules (Massagué, 1990; Ehlers and Riordan, 1991). Like these membrane-anchored growth factors, APP might be active in both membrane-bound and soluble forms, with the interconversion of these forms controlled by a phosphorylation-dependent proteolytic process. In this regard, it is of interest that secreted APP was recently shown to activate mitogen-activated protein kinase, an important component of growth factor-dependent signaling cascades, by a mechanism involving tyrosine phosphorylation (Greenberg et al., 1994). Thus, tyrosine phosphorylation may mediate some of the physiological effects of APPs in addition to regulating its formation.

FOOTNOTES

*

Supported in part by grants from the National Institutes of Health (to R. J. W. and B. E. S.) and the Center for Brain Sciences and Metabolism Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 617-253-8371; Fax: 617-253-6882.

()

The abbreviations used are: APP, amyloid precursor protein; APPs, soluble APP derivatives; PKC, protein kinase C; HEK, human embryonic kidney; PI, phosphoinositide; PMA, phorbol 12-myristate 13-acetate; APLP, APP-like protein.

ACKNOWLEDGEMENTS

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